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J. Zhao, V. Mo, T. Nagasaki; Spatial Distribution of Label–Retaining Cells in the Mouse Limbus . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2094.
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Purpose: Corneal epithelial stem cells are believed to be localized within the basal layer of the limbus, but their precise distribution is not known. This study was initiated to determine the spatial distribution of individual label–retaining cells (LRCs) in the entire circumference of the limbus and also in the cornea. Methods: Mouse pups were injected subcutaneously with 5–bromo–2–deoxy–uridine (BrdU) twice daily for 4 days and were sacrificed after a chase period of 6 to 11 weeks. An eyeball and skinless eyelids were removed together to prepare a globe with continuous ocular surface from the cornea to the mucocutaneous junction. An anterior segment was dissected to prepare a superior and an inferior piece in reference to inner and outer canthus. BrdU immunoreactivity was determined in whole–mount specimens. DAPI nuclear stain was used to differentiate epithelial cells of the cornea and the limbus, and also vertical layers of the epithelium. BrdU positive cells (LRCs) in the cornea and the entire circumference of the limbus were identified under a fluorescent microscope and plotted manually. Since LRCs tended to cluster together, both individual cells and cell clusters were counted and their distribution in different regions of the limbus compared. Limbal blood vessels were identified by CD31 immunostaining. Results: A boundary between the cornea and the limbus could be discerned by nuclear profile of basal epithelial cells; nuclei of peripheral corneal cells were smaller, irregularly shaped, and contained barely detectable nucleoli while limbal cell nuclei were larger, uniformly oval, and exhibited prominent nucleoli. BrdU positive cells were found exclusively in the basal epithelium just outside the cornea–limbus boundary, along the entire limbal circumference, but within a distance of roughly ten cells from the boundary. Most of them were located near the cornea side of the limbal vessel arcade, frequently forming a cluster of several cells. The total number ranged from 200 to 500 per eye. There were about 50%, on average, more cells in the temporal half than the nasal half, and about 50% more in the superior than the inferior, in both right and left eyes. Distribution of LRC clusters, about 150 per eye on average, was similar to that of individual cells. In the cornea, a handful of LRCs were found in the suprabasal layer, but none in the basal layer. Conclusions: Limbal LRCs are present in a narrow zone within the limbus that can be defined precisely in reference to an anatomical boundary between the cornea and the limbus. The results suggest that limbal stem cells are distributed asymmetrically within the limbus. Epithelial stem cells appear to be absent in the cornea proper.
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